Antibacterial Activities of Graphene OxideâMolybdenum Disulfide

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Antibacterial Activities of Graphene OxideMolybdenum Disulfide Nanocomposite Films Tae In Kim, Buki Kwon, Jonghee Yoon, Ick-Joon Park, Gyeong Sook Bang, YongKeun Park, Yeon-Soo Seo, and Sung-Yool Choi ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b12464 • Publication Date (Web): 15 Feb 2017 Downloaded from http://pubs.acs.org on February 15, 2017

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ACS Applied Materials & Interfaces

Antibacterial Activities of Graphene OxideMolybdenum Disulfide Nanocomposite Films Tae In Kim,†,§ Buki Kwon,‡,§ Jonghee Yoon,ǁ Ick-Joon Park,† Gyeong Sook Bang,† YongKeun Park,ǁ,┴ Yeonsoo Seo,‡ and Sung-Yool Choi†*

†

School of Electrical Engineering, Graphene Research Center (GRC), Korea Advanced Institute of Science and Technology (KAIST), Daejeon 34141, Republic of Korea

‡

Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 34141, Republic of Korea ǁ

Department of Physics, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 34141, Republic of Korea ┴

§

TOMOCUBE, Inc., Daejeon 34141, Republic of Korea

T. I. Kim and B. Kwon contributed equally to this work.

* Address correspondence to [email protected].

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ABSTRACT Two-dimensional (2D) nanomaterials, such as graphene-based materials and transition metal dichalcogenide (TMD) nanosheets, are promising materials for biomedical applications owing to their remarkable cytocompatibility and physicochemical properties. Based on their potent antibacterial properties, 2D materials have potential as antibacterial films, wherein the 2D nanosheets are immobilized on the surface and the bacteria may contact with the basal planes of 2D nanosheets dominantly rather than contact with the sharp edges of nanosheets. To address these points, in this study, we prepared an effective antibacterial surface consisting of representative 2D materials, i.e., graphene oxide (GO) and molybdenum disulfide (MoS2), formed into nanosheets on a transparent substrate for real device applications. The antimicrobial properties of the GO-MoS2 nanocomposite surface toward the Gram-negative bacteria Escherichia coli were investigated, and the GO-MoS2 nanocomposite exhibited enhanced antimicrobial effects with increased glutathione oxidation capacity and partial conductivity. Furthermore, direct imaging of continuous morphological destruction in the individual bacterial cells having contacts with the GO-MoS2 nanocomposite surface were characterized by holotomographic (HT) microscopy, which could be used to detect the refractive index (RI) distribution of each voxel in bacterial cell and reconstruct the three-dimensional (3D) mapping images of bacteria. In this regard, both the decreases in the volume (67.2%) and dry mass (78.8%) of bacterial cells came in contact with the surface for 80 min were quantitatively measured, and releasing of intracellular components mediated by membrane and oxidative stress was observed. Our findings provided new insights into the antibacterial properties of 2D nanocomposite film with labelfree tracing of bacterial cell which to improve our understanding of antimicrobial activities, and opened a window for the 2D nanocomposite as a practical antibacterial film in - 2 -


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biomedical applications.

KEYWORDS: antibacterial activity, graphene oxide, molybdenum disulfide, oxidative stress, antibacterial film

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INTRODUCTION Graphene, a two-dimensional (2D) single atomic sheet of sp2-hybridized carbon atoms, has attracted great interest over the past decade due to its extraordinary electrical properties, optical transparency, and biocompatibility.1-5 Graphene oxide (GO) is a waterdispersible graphene sheet with carboxylic, phenol hydroxyl, and epoxide groups on its edges and basal planes, which can be produced by the chemical oxidation of graphite and subsequent exfoliation.6-10 Based on its aqueous stability, low production cost, and amphiphilic behaviors,11-14 GO is a promising material as a building block for graphene-based nanomaterials and their various applications, such as conductive thin film, biosensors, and biomedical devices.15-18 Moreover, molybdenum disulfide (MoS2), a 2D transition metal dichalcogenide (TMD) consisting of hexagonally arranged sulfur atoms linked to a molybdenum atom, has been shown to have potential as a material for biomedical applications due to its intriguing physical and chemical properties.19-20 Various studies of MoS2 have shown that this compound can be used for DNA detection,21 for drug delivery,22 and as a near-infrared photothermal agent owing to its ability to destroy HeLa cells.23 Recently, several groups have reported that 2D materials exert antibacterial effects against various microorganisms through induction of physical damage and oxidative stress.2530

Through direct contact with the bacteria, sharp edges of GO sheet may induce membrane

stress by puncturing or penetrating into the cell membranes, resulting in the morphological destruction of bacterial cells and leakage of intracellular components, such as proteins, phospholipids, RNA, and DNA.24,31,36-38 Moreover, chemical stress is also induced in bacteria by 2D nanosheets that generate the oxidative stress and degrade the cellular structure with or without production of reactive oxygen species (ROS).24,31-35 As a result, both damage to the membrane and leakage of cellular components affected by 2D nanosheets may cause - 4 -


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continuous disruption of the bacterial cells leading to the osmotic imbalance and eventually, the cell death. The antibacterial characteristics of 2D materials, including graphene-based materials and TMD nanosheets, suggest that these materials may have potential as antimicrobial films or surfaces for display devices and biomedical applications, preventing bacterial contamination of the devices without releasing toxic biocides into the environment. However, implementation of antibacterial films consisting of 2D nanosheets for real device applications requires an understanding of the nanotoxicity of the 2D nanosheet film. To this end, antibacterial properties should be investigated in the context of immobilized nanosheets on an appropriate surface, with predominance of antibacterial activity in the basal plane rather than lateral effects of sharp edges. Although many studies have examined the antibacterial properties of GO and MoS2 dispersions,30,34-35 it is unclear whether 2D nanosheet-coated films, e.g., GO-MoS2 nanocomposite surface, could have applications as antimicrobial films for biomedical devices. In this study, we investigated the antibacterial characteristics of antibacterial films consisting of a GO-MoS2 nanocomposite on a transparent substratum toward the Gramnegative bacterium Escherichia coli (E. coli). The probability of superoxide anion (O2•−)induced ROS production was analyzed by the 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2Htetrazolium-5-carboxanilide (XTT) method, and the in vitro γ-L-glutamyl-L-cysteinyl-glycine (glutathione, GSH) oxidation capacity of the 2D nanocomposite was also evaluated. To elucidate the chemical composition of the GO-MoS2 nanocomposite film, Raman spectroscopy and X-ray photoelectron spectroscopy (XPS) were utilized, and scanning electron microscopy (SEM) was used to examine changes in the morphology of bacterial cells. Furthermore, the time evolution of E. coli on the GO-MoS2 nanocomposite film was - 5 -



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analyzed quantitatively using holotomographic (HT) microscopy, which provided threedimensional (3D) refractive index (RI) distribution mapped into tomograms of live bacterial cells in contact with the GO-MoS2 nanocomposite surface.39-42 Changes in the cell volume and dry mass of individual bacterial cells were extracted, and continuous morphological disruption of bacterial cells was demonstrated using 3D RI tomograms. To the best of our knowledge, this study represents the first investigation of the interaction of bacteria with 2D nanosheets surface using label-free tracing analysis with measurements of dry mass and integrity of individual bacterial cells over time.

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MATERIALS AND METHODS Preparation of GO and MoS2 nanosheets. Graphene oxide nanosheets were synthesized by the modified Hummer’s method.52 In the general synthetic process, 1.2 g graphite powder (Sigma Aldrich) was added to a solution containing 15 mL H2SO4, 1 g K2S2O8, and 1 g P2O5 at 80°C with stirring for 4.5 h. The mixture was diluted using distilled water, filtered through a polytetrafluoroethylene (PTFE) membrane filter, and dried in an oven at 60°C for 12 h. Then, 6 g KMnO4 was slowly added to the dried graphite and 46 mL H2SO4 in an ice bath, and the mixture was stirred at 35°C for 2 h and diluted using distilled water. H2O2 was slowly added until the color of the suspension changed from brown to yellow, and after filtering through a PTFE membrane filter, the mixture was redispersed in a 10 wt% HCl solution, followed by filtering. The resulting mixture was dispersed in a 5 wt% H2O2 solution, washed with distilled water, sonicated for exfoliation, and centrifuged at 4000 rpm for 30 min. Finally, the residue was dried and redispersed in distilled water, yielding a 0.1 wt% GO solution. MoS2 nanosheets were prepared from the direct exfoliation of MoS2 powder (< 2 µm, 99%; Sigma Aldrich, St. Louis, MO, USA). In this regard, 8 mg MoS2 was added to 20 mL of 1-methyl-2-pyrrolidinone (NMP, 99%; Sigma Aldrich), a general solvent. The mixture was stirred and sonicated for direct exfoliation using a sonicator (JAC Ultrasonic 2010, 40 kHz, 200 W) with an ice bath system to prevent overheating during sonication. The exfoliated MoS2 solution was centrifuged at 2000 rpm for 2.5 h using a centrifuge (Combi 514R; Hanil Industrial Co., Ltd.) to remove unexfoliated bulk MoS2. The supernatant was sonicated and centrifuged again, and the sediment was then discarded and directly exfoliated MoS2 nanosheets (0.4 mg/mL) were prepared. In thin film deposition process, the cleansed 90-nm SiO2/Si substrate and transparent - 7 -



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silica glass substrate (AMGtech) consisting of SiO2 material were treated by UV-ozone plasma processing using a UV-ozone cleaning system (YUILUV. Co., Ltd ) generating UV emissions at 254 nm (50 mW/cm2) for 300 s to obtain ultra-clean surfaces by removing the surface contaminants of the substrates such as organic contaminants and improve wettability of the surface which results in the excellent adhesion to the substrates and enhancement of stable and uniform deposition of 2D nanosheets, as shown elsewhere.67-68 And GO or MoS2 nanosheet surface was prepared by spin-coating GO or MoS2 nanosheet dispersions five times on the SiO2/Si and silica glass at 2500 rpm for 60 s, respectively. GO-MoS2 nanocomposite surface was prepared by spin-coating GO nanosheet dispersion first and dried in a vacuum desiccator for 24 h to stabilize the nanosheet surface, then followed by spincoating MoS2 nanosheet dispersion under same condition. To remove excess GO or MoS2 nanosheet dispersions, extra spin-coating process without nanosheet dispersion was performed for every nanosheet surfaces, and vacuum drying process was further performed. Characterization. The morphology of the GO and MoS2 nanosheets was analyzed by AFM (PSIA XE-100), and the roughness and height of nanosheets were measured. Raman spectroscopy was utilized for the characterization of the GO and MoS2 nanosheets with irradiation using an Ar ion laser beam at 514 nm with a high-resolution dispersive Raman spectrometer (ARAMIS; Horiba Jobin Yvon, France). The UV-vis spectra of the MoS2 dispersion and transmittance spectra of GO and MoS2 films on the silica glass were obtained with a photodiode array UV-vis spectrophotometer (Scinco S-3100). XPS measurements were conducted using X-ray photoelectron spectroscopy with pass energy of 20 eV and step size of 1 eV. (Sigma Probe, Thermo VG Scientific) Microbial viability test. E. coli K-12 strain of DH5a cells (Enzynomics) were precultured and prepared as shown elsewhere, except the number of bacterial cells used.66 E. coli - 8 -


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cells were diluted with phosphate-buffered saline (PBS; Sigma Aldrich) to 0.5 × 105 CFU/mL and dispensed (0.2 mL) on the film containing GO or MoS2 nanosheets. Bacterial cells dispensed on an empty well were served as a control. To avoid the vaporization of the bacterial cell solution on the surface, wet conditions were used during the microbial viability test. The bacterial cell suspension was harvested after 3-h incubation at 30°C, and the surface of the wafer was washed three times by pipetting with 1 ml of PBS, which was collected as well to harvest any remaining bacterial cells on the surface. All the harvested bacterial cells were centrifuged and resuspended in 1 ml of PBS. Next, 150 µL of the suspension was spread on LB agar plates, and the colonies were counted after overnight incubation at 37°C to estimate the loss of viability of E. coli cells on the GO and MoS2 nanosheet surfaces. All experiments were carried out in triplicate and repeated at least twice. Loss of viability was calculated by the following formula: Loss of viability (%) = (counts of control – counts of samples incubated with 2D nanosheet surfaces)/counts of control ×100 (%). SEM measurements. A field emission SEM (FE-SEM; Magellan400; FEI Company) was used to observe morphological changes in bacteria interacting with the GO-MoS2 nanocomposite surface. 0.5 × 105 CFU/mL of bacterial cells were dispensed on the GO-MoS2 nanocomposite surface for 3 h at 30°C and the fixation of bacterial cells was carried out using 2.5% glutaraldehyde, and bacterial cells were then washed with PBS and dehydrated through a graded ethanol series (30, 50, 70, and 100 v/v%) for 15 min. For SEM measurement, bacterial cells were coated with osmium, and a 3 kV electron beam with magnification of 104 or 2×104 was used to observe the destruction of bacterial morphology. Oxidative stress measurements. To analyze the possibility of superoxide radical anion (O2•−) generation by the GO and MoS2 nanosheets, the absorption of 2,3-bis-(2methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT; Fluka) was measured - 9 -



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using a UV-vis spectrophotometer (Scinco S-3100). One milliliter of GO or MoS2 dispersion in PBS was added to 1 mL XTT solution in PBS (0.4 mM); XTT solution without nanosheets was used as a control sample. The mixture was incubated in the dark for 2, 4, or 6 h with shaking, and GO or MoS2 nanosheets were removed using a 0.25-µm syringe filter. The filtrates were transferred to the UV-vis well to measure the absorbance; XTT can be reduced by the superoxide radical anion forming water-soluble XTT-formazan, which shows maximum absorbance at 470 nm.51 To investigate the ROS-independent oxidative stress pathway induced by GO and MoS2 nanosheets, GSH oxidation was measured by Ellman’s assay.49 Briefly, 0.5 mL GO or MoS2 dispersion in 50 mM bicarbonate buffer (pH 8.6) was added to 0.5 mL GSH solution in bicarbonate buffer (0.8 mM), and the mixture was incubated in the dark for 2, 4, or 6 h with shaking. H2O2 (1 mM) was added to the GSH solution (0.4 mM) as a positive control. After incubation, 50 mM Tris-HCl (1.5 mL) and 100 mM DNTB solution (30 µL) were added to the mixture, and the mixture was filtered using 0.25-µm syringe filter. The filtrates were transferred to the UV-vis well to measure the absorbance in order to detect GSH oxidation, and the oxidation was estimated by comparing the absorbance at 412 nm with the negative control (GSH solution without nanosheets) using the following equation: loss of GSH (%) = (absorbance of negative control – absorbance of sample) / (absorbance of negative control) × 100 (%). All samples were tested in triplicate. HT Analysis. To measure the 3D RI distribution of individual bacterial cells, the bacteria were placed on the transparent GO-MoS2 nanocomposite surface and then covered with a bare cover glass to prevent dehydration. The 3D RI tomograms of bacteria were obtained using a 3D holotomographic (HT) microscopy (HT-1; TOMOCUBE Inc., Republic of Korea), where HT microscopy can detect the phase difference of light coming through the - 10 -


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bacterial cells and reconstruct the 3D RI image, a tomogram of bacteria.39-40 Typically, 101 2D optical field images of samples were measured, from which a 3D RI tomogram was reconstructed via the optical diffraction tomography algorithm. The volume of a single bacterial cell was obtained by integrating voxels having higher RI values than that of surrounding medium, and the dry mass of the bacterium was provided by 3D RI values because RI values are linearly proportional to the concentration of non-aqueous molecules as shown elsewhere,42-44 and all measurements were repeated at least twenty times. The details of optical field retrieval and tomogram reconstruction processes can be found elsewhere.41-42

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RESULTS AND DISCUSSION Preparation and Characterization of GO, MoS2, and GO-MoS2 Nanocomposite Surfaces. GO nanosheets were produced by the chemical oxidation of graphite and exfoliation using a modified Hummer’s method.52 MoS2 nanosheets were prepared by chemical-assisted direct exfoliation of bulk MoS2 powder including ultrasonication and filtering process. Each nanosheet surface was prepared as described in the Materials and Methods. Figure 1A shows a schematic of the GO and MoS2 nanosheet surfaces and the GO-MoS2 nanocomposite surface on the 90-nm SiO2/Si substrate. Atomic force microscopy (AFM) images of GO, MoS2, and the GO-MoS2 nanocomposite surfaces are shown in Figure 1B–D, demonstrating the topographs and surface characteristics of nanosheet films. The average roughness values of GO nanosheet surface, MoS2 nanosheet surface, and the GO-MoS2 nanocomposite surface are 0.9 nm, 0.5 nm, and 1.2 nm, respectively, showing that the surfaces of the nanosheet films are uniform and the roughness increases as GO and MoS2 nanosheets are formed into GOMoS2 nanocomposite surface. The average thickness of the GO nanosheet was 1.2 nm, indicating that the GO nanosheets were formed in a single layer, whereas that of MoS2 nanosheet was 2.2 nm which exhibits the multilayer MoS2 nanosheet. (see insets in Figure 1B–D). From the topographs and thickness of nanosheets in AFM images, we confirmed the orientation of nanosheets with respect to the surface in which nanosheets were placed on the surface in the horizontal direction as the basal planes of nanosheets rather than sharp edges were exposed on the surface. The functionalization of GO, MoS2, and GO-MoS2 nanocomposite film was characterized by Raman spectroscopy, as shown in Figure S1A-C. The G band centered at 1590 cm-1 in Raman spectrum of GO nanosheet surface corresponds to the sp2-graphitized - 12 -


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structure of carbon while the D band positioned at 1350 cm-1 is attributed to the defects or disorders existing in the crystalline structure, which reveals the oxidation of the graphitized structure as shown in Figure S1A. In the Raman spectrum of GO-MoS2 nanocomposite surface, the G band and D band were also observed (Figure S1C), however the intensities of the G band and D band were different from those of GO nanosheet surface, in which the intensity ratio of the D band to the G band (ID/IG) was 1.01 for the GO-MoS2 nanocomposite surface and 1.28 for the GO nanosheet surface (Figure S1D). This decrease in the intensity of D band normalized by the intensity of G band indicated the improvement in the sp2graphitized structure of GO-MoS2 nanocomposite surface containing reduced defects or disorders caused by partial reduction of GO combined with MoS2 nanosheets.27 The Raman spectrum of the MoS2 nanosheet surface exhibits the two most intense peaks that are related to the vibrational modes of the E12g peak at 380.4 cm-1 and the A1g peak at 405.4 cm-1 in Figure S1B. The wavenumber difference between the E12g peak and A1g peak (around 25 cm-1) indicates that the MoS2 nanosheets are multilayered.45 Figure S1C depicts the Raman spectra of the GO-MoS2 nanocomposite surface, clearly showing peaks generated from GO (D and G peaks) and MoS2 nanosheets (E12g peak and A1g peak). The MoS2 nanosheets were further characterized by UV-vis spectroscopy (Figure S2). The two peak positions for the excitonic A band at 670 nm and excitonic B band at 610 nm demonstrated the 2H-phase MoS2 corresponding to the direct transition from the valence band to the conduction band, consistent with previous studies.46-48 The semiconducting 2H-phase MoS2 nanosheets rather than metallic 1T-phase were also confirmed by the Mo 3d and S 2p peaks from the XPS measurements (Figure S3). Antibacterial Activity of GO, MoS2, and GO-MoS2 Nanocomposite Surfaces. Next, we investigated the antibacterial activities of GO, MoS2, and the GO-MoS2 - 13 -



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nanocomposite film. As a bacterial cell model, E. coli K-12 strain of DH5α cells (0.5 × 105 CFU/mL) were dispensed on GO, MoS2, and the GO-MoS2 nanocomposite surface and incubated for 3 h. E. coli cells incubated without nanocomposite surface were used as a control. As a result of direct contact between bacteria and 2D nanomaterial film, bacterial cells were inactivated and lost their viabilities in agreement with the antibacterial activities of carbon-based materials, e.g., fullerene,53 carbon nanotubes (CNTs),54-57 and GO,27,30-32 where their contact-based antibacterial activities may generate the physical disruption and direct oxidation of intracellular components.53-54,57-58 As shown in Figure 2A, the significant differences between the loss of bacterial viability cultured on GO, MoS2, and GO-MoS2 nanocomposite film were demonstrated (p < 0.05) where the loss of E. coli viability was higher for bacterial cells cultured on the GOMoS2 nanocomposite film than that of bacterial cells cultured on either GO or MoS2 nanosheet surfaces, indicating that GO-MoS2 nanocomposite surface exhibited synergistic effects as having enhanced antimicrobial capacities toward bacterial cells. Notably, under our concentration conditions, the nanotoxicity of MoS2 nanosheet surface was higher than that of GO nanosheet surface. Unlike the antibacterial effects of GO and MoS2 nanosheets in suspension assays, in which bacterial cells may interact with sharp edges of nanosheets in every direction, our GO-MoS2 nanocomposite film has limited interactions with bacterial cells primarily on the basal planes along the surface. Nevertheless, approximately 61% of the bacterial cells were inactivated in comparison with the control bacterial cells owing to interactions with the GO-MoS2 nanocomposite surface only for 2 h, and this value increased to 80% after incubation for 4 h, and almost every bacterial cells were destroyed (96.4%) after incubation for 6 h (Figure 2B) as showing the statistical significance (p < 0.05) along the incubation time. From the cell inactivation results, our nanocomposite film showed the - 14 -


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effective antibacterial capacities having great potentials as a practical antimicrobial film for biomedical applications. These results support the previous results on the important role of 2D basal planes rather than edges in antimicrobial mechanisms, in which completely flat Langmuir-Blodgett films revealed the antimicrobial property toward bacterial cells having few contacts with sheet edges.59-60 The morphology of E. coli cells was evaluated by scanning electron microscopy (SEM) measurements to investigate the destruction of the cell membrane (Figure 2C–F). In Figure 2C and 2E, SEM images of control bacterial cells are demonstrated at magnifications of 104 and 2×104, respectively, showing rod-shaped morphologies of E. coli bacteria. As shown in Figure 2D and 2F, morphologies of bacterial cells came in contact with the GOMoS2 nanocomposite surface, and importantly, severely damaged cell membranes with holes in the cellular surface were observed after 3-h incubation on the GO-MoS2 nanocomposite surface. Moreover, cytoplasmic leakage and intracellular components were also observed outside the bacterial cells, indicating that direct contact with nanocomposite surface generated serious destruction to the cell membranes leading to the cell death. Additional SEM images of bacteria with compromised cell integrity after having contacts with 2D nanocomposite surface can be found in the Supporting Information. The SEM images provide evidence for major morphological damage owing to the antibacterial activities of the 2D nanocomposite film that may generate the direct physical disruption, chemical oxidative stress, and charge transfer from the bacterial cells to the nanosheets. Oxidative Stress Mediated Antimicrobial Property. Previous studies have proposed several antibacterial mechanisms of 2D materials, including the physical interaction between bacterial cells and GO nanosheets and chemical oxidation of intracellular components. Through direct contact with the bacteria, GO sheets - 15 -



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may act as a sharp blade that gives the physical stress by piercing or penetrating into the cell membranes. From the molecular dynamics simulations, it is revealed that the penetration of GO nanosheets results in the extraction of the phospholipid bilayers and degradation of the inner and outer cell membranes.31 The cell entrapment of GO nanosheets is also proposed to inactivate the bacterial cells that may cause the isolation of bacteria from the environment.61 In addition to membrane stress-mediated physical interaction, chemical oxidative stress is an important issue affecting the antibacterial activity that induces the oxidation of intracellular components such as proteins, phospholipids, and nucleic acids.30-35 The oxidation capacity is mainly dependent on the physicochemical property of the nanomaterials, i.e., size of nanosheet, oxidation level, or functionalized structure. In graphene-based materials, it is reported that the oxidation capacity of chemically reduced GO is higher than that of GO,30 and it is also shown that GO has different oxidation capacity due to its size of nanosheet.62 For CNTs, single-walled carbon nanotubes (SWNTs) was observed to generate higher oxidative stress as a fraction of metallic SWNTs increases.54 Thus, the oxidative stress generated by GO-MoS2 nanocomposite surface was investigated focusing on the analysis of physicochemical property. The oxidative stress generated by 2D nanomaterials may originate from different pathways. For example, ROS-dependent oxidative stress, in which the superoxide anion (O2•−) generated by nanomaterials induces oxidative stress and disrupts microbial viability, and ROS-independent oxidative stress, in which the nanomaterials destroy a specific cellular process by oxidizing a vital microbial structure or cellular component without any ROS production, have both been observed.50 Therefore, to investigate the antibacterial mechanisms of the GO-MoS2 nanocomposite, ROS-dependent and -independent oxidative stress pathways were measured by XTT and GSH test, respectively. As shown in Figure 3A, XTT result - 16 -


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revealed the significant differences between the ROS production capacities of GO, MoS2, and GO-MoS2 nanocomposite (p < 0.05) where ROS production increased after 4-h incubation with MoS2 nanosheets; in contrast, the GO nanosheets did not produce any superoxide anions as compared with the control. Interestingly, the GO-MoS2 nanocomposite produced superoxide anion; however, the absorbance was lower than that of MoS2 nanosheets, implying that ROS production by the MoS2 nanosheets was inhibited. Although enhanced antibacterial properties were observed in the GO-MoS2 nanocomposite film from the microbial viability test, our XTT test results indicate that ROS-dependent oxidative stress may play a minor role in the antibacterial mechanism of the GO-MoS2 nanocomposite. Next, GSH oxidation was detected to determine whether ROS-independent oxidative stress was generated by GO and MoS2 nanosheets using Ellman’s assays.49 GSH, a tripeptide with thiol groups, functions as an antioxidant in bacteria at a concentration of 0.1 to 10 mM by oxidizing thiol groups (–SH) to disulfide bonds (–S–S–) and it is converted to glutathione disulfide to prevent cellular damage caused by oxidative stress.50 As shown in Figure 3B, both GO and MoS2 nanosheets induced GSH oxidation and the significant differences between the GSH oxidation capacities of GO, MoS2, and GO-MoS2 nanocomposite (p < 0.05) were demonstrated where GO nanosheets showed higher oxidation capacity (29.5% ± 0.3%, 54% ± 0.5%, and 50.3% for 2, 4, and 6 h, respectively) than the MoS2 nanosheets (26.2%, 41.1% ± 0.6%, and 40.2% ± 0.3% for 2, 4, and 6 h, respectively). The amount of oxidation increased during the exposure time of 4 h and slightly decreased after incubation for 6 h in all samples. Owing to the synergistic effects of GO and MoS2 naonosheets, the GO-MoS2 nanocomposite showed enhanced oxidation capacity (56.2% ± 0.3%, 67.8%, and 67.6% ± 0.3% for 2, 4, and 6 h, respectively), where partial reduction of GO in the GO-MoS2 nanocomposite may induce GSH oxidation to a greater extent than the GO nanosheet itself - 17 -



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due to the higher conductivity of the nanocomposite.30 Thus, these findings regarding GSH oxidation imply that the GO-MoS2 nanocomposite is capable of generating ROS-independent oxidative stress toward bacterial cells and that ROS-independent stress, such as charge transfer from the cell membrane to the nanosheets, may be a strong antibacterial mechanism of the GO-MoS2 nanocomposite. From XTT and GSH results shown in Figure 3, the different ROS-dependent and -independent oxidation capacities of three different nanosheets were investigated, and the antibacterial mechanisms of GO, MoS2, and GO-MoS2 nanocomposite were compared in which GO nanosheet showed only GSH oxidation capacity while MoS2 nanosheet showed both ROS-dependent oxidation and GSH oxidation capacity, and in case of GO-MoS2 nanocomposite, the synergistic oxidation capacities including ROS-dependent oxidation and GSH oxidation capacity were revealed leading to the enhanced antibacterial properties. For further investigation of antibacterial mechanisms of nanomaterials, the functional groups and chemical states of GO nanosheet, MoS2 nanosheet, and GO-MoS2 nanocomposite surface were characterized by high-resolution XPS analysis (Figure 4). The deconvoluted C(1s) XPS spectrum of GO is shown in Figure 4A, and that of the GO-MoS2 nanocomposite is shown in Figure 4B. A binding energy of 285 eV indicates C-C, C=C, and C-H bonds in GO nanosheets, while that of 286.1 eV is attributed to the C-OH functional group. The C=O and O=C-OH functional groups on the surface of GO can be detected at binding energies of 287.4 and 289 eV.27 In Figure 4C, the intensity ratios of C-OH, C=O, and O=C-OH groups to C-C, C=C, and C-H bonds in the GO nanosheet and GO-MoS2 nanocomposite film were investigated, where the intensities of C-OH, C=O, and O=C-OH groups normalized by C-C, C=C, and C-H bonds in the GO decreased after GO and MoS2 nanosheets were formed into GO-MoS2 nanocomposite surface, indicating the different composition of functional groups - 18 -


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and chemical structures between GO nanosheet and GO-MoS2 nanocomposite surface. This result implies that the oxygen-based functional groups of the sp2-graphitized structure in GO are partially reduced when GO is combined with MoS2 nanosheets yielding a GO-MoS2 nanocomposite film with relatively higher conductivity than GO nanosheets. In regard to the charge transfer mechanism, the GO-MoS2 nanocomposite may promote charge transport from the cell membrane to the nanocomposite, causing disruption of intracellular components through oxidation, which can lead to a cell death. Our structural analysis of the nanocomposite is consistent with the enhanced GSH oxidation capacity of GO-MoS2 nanocomposite shown in Figure 3B. The XPS spectra of Mo peak and S peak in the MoS2 nanosheets and the GO-MoS2 nanocomposite (Figure S3) showed that Mo peaks centered at 232 and 229 eV corresponded to Mo 3d3/2 and Mo 3d5/2, respectively, and that S peaks centered at 163 and 162 eV resulted from the S 2p1/2 and S 2p3/2 orbitals, respectively, implying the occurrence of 2H-phase MoS2 rather than 1T-phase MoS2 in both MoS2 nanosheets and the GO-MoS2 nanocomposite film.35 Continuous morphological destruction of bacterial cells came in contact with transparent antimicrobial film by real-time 3D analysis. To implement the practical antimicrobial surface for the biomedical device applications, the GO-MoS2 nanocomposite film was prepared on the transparent silica glass substrate (see the Materials and Methods). The GO-MoS2 nanocomposite surface on the silica glass was characterized by AFM, as shown in Figure S5; the average roughness of the GOMoS2 nanocomposite surface was 1 nm, whereas that of bare silica glass was 0.4 nm. These are similar to the roughness of the GO-MoS2 nanocomposite surface on the SiO2/Si and bare SiO2/Si where the average roughness of GO-MoS2 nanocomposite surface on the SiO2/Si is 1.2 nm and that of bare SiO2/Si is 0.4 nm. Since the silica glass and SiO2/Si surfaces - 19 -



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exhibited the similar level of roughness while consisting of same material, it was confirmed that GO-MoS2 nanocomposite film was well deposited on the silica glass showing the surface condition analogous to the SiO2/Si. Therefore, the antibacterial activity of GO-MoS2 nanocomposite surface on silica glass toward E. coli bacterial cells can be evaluated and compared to the 2D nanocomposite surface on SiO2/Si as shown in Figure 5A. After exposure for 3 h, the loss of E. coli viability was 58.6% for the GO-MoS2 nanocomposite surface on the silica glass and 61.1% for SiO2/Si, respectively, indicating that no significant differences (p > 0.05) between the antibacterial properties of GO-MoS2 nanocomposite surface on the silica glass and SiO2/Si could be found. Based on the potential of the transparent antibacterial 2D nanocomposite film for biomedical applications, we next measured the transmittance of GO-MoS2 nanocomposite film on a glass and bare glass by UV-vis spectrophotometry, as shown in Figure 5B. At a wavelength of 550 nm, the transmittance of the antibacterial film was around 99%. Additionally, the transparent antibacterial film on the glass could not be distinguished from the bare glass. Therefore, our results demonstrate that the transparent antibacterial film consisting of the GO-MoS2 nanocomposite has excellent antimicrobial properties and can be used for biomedical applications as an antibacterial surface. There are various methods for investigating the antibacterial effects of 2D nanomaterials, such as colony counting method which is generally used to verify the viability of bacteria, SEM measurement to observe the morphology of bacterial cell, and live/dead fluorescent staining method by using impermeable propidium iodide dye where the viability of cell is determined by the green or red color under confocal laser scanning microscopy.62 However, these are limited in tracing the bacterial cells came in contact with the 2D nanomaterials over time, and observing continuous change in bacterial membrane for detailed study on the antibacterial mechanism of 2D nanomaterials. To further investigate the - 20 -


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antibacterial effects of the GO-MoS2 nanocomposite film and monitor the morphological change of bacteria without fluorescent staining-based labeling in real time, the 3D RI tomograms of individual live bacterial cells were therefore measured with the HT microscopy every 30 s for 90 min. Figure 6A shows representative cross-sectional slices of 3D RI tomograms of bacteria along the x-z and x-y axes after incubation for 0, 30, 60, and 90 min. The RI distribution of the tomogram ranging from 1.34 to 1.41 clearly separates the bacteria from the background medium, and cell components inside the bacteria are well demonstrated by different color having the appropriate RI values of E. coli bacteria proposed in the previous studies.63-65 Although the cellular components of bacteria, e.g., cell wall, membrane, proteins, or DNA, cannot be completely distinguishable due to the lack of information in accurate RI values of bacterial components, but the 3D RI tomograms provide separation between cell membrane and materials such as cytoplasm and DNA, obviously. In Figure 6B, corresponding 3D rendered images of the bacteria along x-y and y-z axes are obtained via mapping the RI distribution of bacterial cells into 3D images. The bacteria exhibited a rodshape after loading on the GO-MoS2 nanocomposite film; however, morphological damage was gradually observed in the bacterial membrane, with obvious disruption of the bacteria after 60 min. These HT results are consistent with the SEM images, in which the cellular structures of bacteria are found to be damaged as a result of the antimicrobial activity of the GO-MoS2 nanocomposite. Continuous morphological destruction of bacterial cells is shown in Supporting Movie S1. To quantify the antibacterial effects of the GO-MoS2 nanocomposite film, the volume and dry mass of individual bacteria were further calculated from the measured 3D RI tomograms. Figure 6C and 6D show the relative dry mass and bacterial cell volume over time. The relative dry masses of damaged bacteria after 20 or 80 min of incubation on the GO- 21 -



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MoS2 nanocomposite film were 93.3% ± 13% and 78.8% ± 12.1%, respectively, whereas the dry mass of live bacteria did not change substantially. The relative cell volumes of damaged bacteria were 74.5% ± 12.3% and 67.2% ± 13% after 20 or 80 min of incubation, respectively. The significant differences of dry masses and cell volumes between live bacteria and damaged bacteria are shown, and the asterisks indicate the statistical significance with different number of asterisks showing statistical differences (p < 0.05) in Figure 6C and 6D. These results clearly indicate that the GO-MoS2 nanocomposite film decreases both bacterial volume and dry mass, accompanied by loss of intracellular components and cell membranes as a result of the physical destruction and oxidative stress generated from the 2D nanocomposite. Therefore, the antibacterial activity of 2D nanocomposite surface with immobilized nanosheets has been investigated by using various tools. The oxidative stress has been shown as a dominant mechanism that kills the bacteria where the action of the basal planes of 2D nanocomposite surface is an important factor to the antibacterial properties of 2D nanocomposite surface rather than sharp edges based physical action of nanosheets. The oxidation of intracellular components was revealed by label-free tracing of bacteria over time, and loss of cellular materials was quantitatively measured. Our results represent the effective transparent antibacterial film consisting of 2D nanocomposite, and open a great potential of 2D nanocomposite film as practical biomedical device applications. The intimate interaction between 2D nanocomposite and bacterial cell along the oxidative pathways needs to be further investigated to verify the antibacterial mechanism of 2D nanocomposite in details.

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CONCLUSION Antibacterial surfaces consisting of GO, MoS2, and the GO-MoS2 nanocomposite were evaluated and their antimicrobial properties toward E. coli were investigated. Colony counting assays revealed the enhanced antibacterial properties of GO-MoS2 nanocomposite film, which could be explained by the increased oxidation capacity and conductivity of the GO-MoS2 nanocomposite film due to the partially reduced GO in combination with MoS2 nanosheets. The measured oxidative stress showed that the GO-MoS2 nanocomposite had higher ROS-independent oxidation capacity, confirming that GSH oxidative stress was a major antibacterial mechanism of the GO-MoS2 nanocomposite. However, the ROSdependent oxidative stress induced by GO-MoS2 nanocomposite was found to play a minor role in the antibacterial mechanism showing the lower superoxide radical production than MoS2 nanosheets. Furthermore, the decreases in the cell volume and dry mass of individual bacterial cells were quantitatively investigated by using 3D real-time HT microscopy, which enables label-free tracing of bacteria in contact with nanocomposite surface and generates the RI distribution of bacteria. The reduced cell volume and dry mass imply the oxidation of cell structure and leakage of intracellular components through holes on the cell membrane that are consistent with our GSH results and SEM micrographs. These results provide quantitative information regarding the antibacterial effects of the GO-MoS2 nanocomposite film over time, therefore suggest new insights into the potential biomedical applications of the GO-MoS2 nanocomposite film as a transparent antibacterial film.

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Conflict of Interests The authors declare no competing financial interests.

Acknowledgments This work was supported by the Creative Materials Discovery Program of the National Research Foundation of Korea (NRF) (2016M3D1A1900035), the Nano-Material Technology Development Program of NRF (2012M3A7B4049807), and LG Display Co., Ltd.

Supporting Information Available Representative Raman spectra of nanosheets and the intensity ratio of the D band to G band (ID/IG); UV-vis spectrum of 2H-phase MoS2 nanosheets; X-ray photoelectron spectroscopy (XPS) spectra of Mo and S peaks in MoS2 and the GO-MoS2 nanocomposite; scanning electron microscopy (SEM) images of control bacteria and bacteria came in contact with GOMoS2 nanocomposite surface; and atomic force microscopy (AFM) images of the GO-MoS2 nanocomposite film on the SiO2/Si and silica glass; Label-free tracing by holotomographic (HT) microscopy : E. coli. This material is available free of charge on the ACS Publications website at http://pubs.acs.org.

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REFERENCES AND NOTES 1. Geim, A. K.; Novoselov, K. S. The Rise of Graphene. Nat. Mater. 2007, 6, 183–191. 2. Novoselov, K. S.; Geim, A. K.; Morozov, S. V.; Jiang, D.; Zhang, Y.; Dubonos, S. V.; Grigorieva, I. V.; Firsov, A. A. Electric Field Effect in Atomically Thin Carbon Films. Science 2004, 306, 666–669. 3. Geim, A. K. Graphene: Status and Prospects. Science 2009, 324, 1530–1534. 4. Novoselov, K. S.; Geim, A. K.; Morozov, S. V.; Jiang, D.; Katsnelson, M. I.; Grigorieva, I. V.; Dubonos, S. V.; Firsov, A. A. Two-Dimensional Gas of Massless Dirac Fermions in Graphene. Nature 2005, 438, 197–200. 5. Bitounis, D.; Ali-Boucetta, H.; Hong, B. H.; Min, D.-H.; Kostarelos, K. Prospects and Challenges of Graphene in Biomedical Applications. Adv. Mater. 2013, 25, 2258–2268. 6. Dreyer, D. R.; Park, S.; Bielawski, C. W.; Ruoff, R. S. The Chemistry of Graphene Oxide. Chem. Soc. Rev. 2010, 39, 228–240. 7. Park, S.; Ruoff, R. S. Chemical Methods for the Production of Graphenes. Nat. Nanotechnol. 2009, 4, 217–224. 8. Compton, O. C.; Nguyen, S. T. Graphene Oxide, Highly Reduced Graphene Oxide, and Graphene: Versatile Building Blocks for Carbon-Based Materials. Small 2010, 6, 711–723. 9. Zhu, Y.; Murali, S.; Cai, W.; Li, X.; Suk, J. W.; Potts, J. R.; Ruoff, R. S. Graphene and Graphene Oxide: Synthesis, Properties, and Applications. Adv. Mater. 2010, 22, 3906–3924. 10. Paredes, J. I.; Villar-Rodil, S.; Martinez-Alonso, A.; Tascon, J. M. D. Graphene Oxide Dispersions in Organic Solvents. Langmuir 2008, 24, 10560–10564. - 25 -



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

11. Li, D.; Muller, M. B.; Gilje, S.; Kaner, R. B.; Wallace, G. G. Processable Aqueous Dispersions of Graphene Nanosheets. Nat. Nanotechnol. 2008, 3, 101–105. 12. Cote, L. J.; Kim, F.; Huang, J. X. Langmuir-Blodgett Assembly of Graphite Oxide Single Layers. J. Am. Chem. Soc. 2009, 131, 1043–1049. 13. Kim, F.; Cote, L. J.; Huang, J. X. Graphene Oxide: Surface Activity and TwoDimensional Assembly. Adv. Mater. 2010, 22, 1954–1958. 14. Cote, L. J.; Kim, J.; Tung, V. C.; Luo, J. Y.; Kim, F.; Huang, J. X. Graphene Oxide as Surfactant Sheets. Pure Appl. Chem. 2011, 83, 95–110. 15. Wang, Y.; Li, Z. H.; Wang, J.; Li, J. H.; Lin, Y. H. Graphene and Graphene Oxide: Biofunctionalization and Applications in Biotechnology. Trends Biotechnol. 2011, 29, 205–212. 16. Kuila, T.; Bose, S.; Khanra, P.; Mishra, A. K.; Kim, N. H.; Lee, J. H. Recent Advances in Graphene-Based Biosensors. Biosens. Bioelectron. 2011, 26, 4637–4648. 17. Feng, L. Z.; Liu, Z. A. Graphene in Biomedicine: Opportunities and Challenges. Nanomedicine 2011, 6, 317–324. 18. Chen, F. M.; Liu, S. B.; Shen, J. M.; Wei, L.; Liu, A. D.; Chan-Park, M. B.; Chen, Y. Ethanol-Assisted Graphene Oxide-Based Thin Film Formation at Pentane-Water Interface. Langmuir 2011, 27, 9174–9181. 19. Mak, K. F.; Lee, C.; Hone, J.; Shan, J.; Heinz, T. F. Atomically Thin MoS2: A New Direct-Gap Semiconductor. Phys. Rev. Lett. 2010, 105, 136805. 20. Xu, M. S.; Liang, T.; Shi, M. M.; Chen, H. Z. Graphene-Like Two-Dimensional Materials. Chem. Rev. 2013, 113, 3766–3798. 21. Zhu, C.; Zeng, Z.; Li, H.; Li, F.; Fan, C.; Zhang, H. Single-Layer MoS2-Based Nanoprobes for Homogeneous Detection of Biomolecules. J. Am. Chem. Soc. 2013, - 26 -


Page 26 of 41

Page 27 of 41

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


135, 5998–6001. 22. Liu, T.; Wang, C.; Gu, X.; Gong, H.; Cheng, L.; Shi, X. Z.; Feng, L. Z.; Sun, B. Q.; Liu, Z. Drug Delivery with PEGylated MoS2 Nano-Sheets for Combined Photothermal and Chemotherapy of Cancer. Adv. Mater. 2014, 26, 3433–3440. 23. Chou, S. S.; Kaehr, B.; Kim, J.; Foley, B. M.; De, M.; Hopkins, P. E.; Huang, J. X.; Brinker, C. J.; Dravid, V. P. Chemically Exfoliated MoS2 as Near-Infrared Photothermal Agents. Angew. Chem. Int. Ed. 2013, 52, 4254–4258. 24. Krishnamoorthy, K.; Veerapandian, M.; Zhang, L.; Yun, K.; Kim, S. J. Antibacterial Efficiency of Graphene Nanosheets against Pathogenic Bacteria via Lipid Peroxidation. J. Phys. Chem. C 2012, 116, 17280–17287. 25. Li, J.; Wang, G.; Zhu, H.; Zhang, M.; Zheng, X.; Di, Z.; Liu, X.; Wang, X. Antibacterial Activity of Large-Area Monolayer Graphene Film Manipulated by Charge Transfer. Sci. Rep. 2014, 4, 4359. 26. Hu, W. B.; Peng, C.; Luo, W. J.; Lv, M.; Li, X. M.; Li, D.; Huang, Q.; Fan, C. H. Graphene-Based Antibacterial Paper. ACS Nano 2010, 4, 4317–4323. 27. Akhavan, O.; Ghaderi, E. Toxicity of Graphene and Graphene Oxide Nanowalls against Bacteria. ACS Nano 2010, 4, 5731–5736. 28. Sanchez, V. C.; Jachak, A.; Hurt, R. H.; Kane, A. B. Biological Interactions of Graphene-Family Nanomaterials: An Interdisciplinary Review. Chem. Res. Toxicol. 2012, 25, 15–34. 29. Chen, J.; Peng, H.; Wang, X.; Shao, F.; Yuan, Z.; Han, H. Graphene Oxide Exhibits Broad-Spectrum Antimicrobial Activity against Bacterial Phytopathogens and Fungal Conidia by Intertwining and Membrane Perturbation. Nanoscale 2014, 6, 1879–1889. 30. Liu, S.; Zeng, T. H.; Hofmann, M.; Burcombe, E.; Wei, J.; Jiang, R.; Kong, J.; Chen, - 27 -



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Y. Antibacterial Activity of Graphite, Graphite Oxide, Graphene Oxide, and Reduced Graphene Oxide: Membrane and Oxidative stress. ACS Nano 2011, 5, 6971–6980. 31. Tu, Y.; Lv, M.; Xiu, P.; Huynh, T.; Zhang, M.; Castelli, M.; Liu, Z.; Huang, Q.; Fan, C.; Fang, H.; Zhou, R. Destructive Extraction of Phospholipids from Escherichia Coli Membranes by Graphene Nanosheets. Nat. Nanotechnol. 2013, 8, 594–601. 32. Gurunathan, S.; Han, J. W.; Dayem, A. A.; Eppakayala, V.; Kim, J.-H. Oxidative Stress-Mediated Antibacterial Activity of Graphene Oxide and Reduced Graphene Oxide in Pseudomonas Aeruginosa. Int. J. Nanomed. 2012, 7, 5901–5914. 33. Navale, G. R.; Rout, C. S.; Gohil, K. N.; Dharne, M. S.; Late, D. J.; Shinde, S. S. Oxidative and Membrane Stress-Mediated Antibacterial Activity of WS2 and rGOWS2 Nanosheets. RSC Adv. 2015, 5, 74726–74733. 34. Liu, S.; Hu, M.; Zeng, T. H.; Wu, R.; Jiang, R.; Wei, J.; Wang, L.; Kong, J.; Chen, Y. Lateral Dimension-Dependent Antibacterial Activity of Graphene Oxide Sheets. Langmuir 2012, 28, 12364–12372. 35. Yang, X.; Li, J.; Liang, T.; Ma, C.; Zhang, Y.; Chen, H.; Hanagata, N.; Su, H.; Xu, M. Antibacterial Activity of Two-Dimensional MoS2 Sheets. Nanoscale 2014, 6, 10126– 10133. 36. He, S.; Song, B.; Li, D.; Zhu, C.; Qi, W.; Wen, Y.; Wang, L.; Song, S.; Fang, H.; Fan, C. A Graphene Nanoprobe for Rapid, Sensitive, and Multicolor Fluorescent DNA Analysis. Adv. Funct. Mater. 2010, 20, 453–459. 37. Lv, W.; Guo, M.; Liang, M.-H.; Jin, F.-M.; Cui, L.; Zhi, L.; Yang, Q.-H. GrapheneDNA Hybrids: Self-Assembly and Electrochemical Detection Performance. J. Mater. Chem. 2010, 20, 6668–6673. 38. Alava, T.; Mann, J. A.; Theodore, C.; Benitez, J. J.; Dichtel, W. R.; Parpia, J. M.; - 28 -


Page 28 of 41

Page 29 of 41

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Craighead, H. G. Control of the Graphene-Protein Interface Is Required To Preserve Adsorbed Protein Function. Anal. Chem. 2013, 85, 2754–2759. 39. Shin, S.; Kim, K.; Yoon, J.; Park, Y. Active Illumination Using a Digital Micromirror Device for Quantitative Phase Imaging. Opt. Lett. 2015, 40, 5407–5410. 40. Shin, S.; Kim, K.; Kim, T.; Yoon, J.; Hong, K.; Park, J.; Park, Y. Optical Diffraction Tomography Using a Digital Micromirror Device for Stable Measurements of 4D Refractive Index Tomography of Cells. In Quantitative Phase Imaging II, SPIE BiOS, International Society for Optics and Photonics 2016, 971814. 41. Kim, K.; Yoon, H.; Diez-Silva, M.; Dao, M.; Dasari, R. R.; Park, Y. High-Resolution Three-Dimensional Imaging of Red Blood Cells Parasitized by Plasmodium Falciparum and in Situ Hemozoin Crystals Using Optical Diffraction Tomography. J. Biomed. Opt. 2015, 19, 011005. 42. Kim, K.; Yoon, J.; Shin, S.; Lee, S.; Yang, S.-A.; Park. Y. Optical Diffraction Tomography Techniques for the Study of Cell Pathophysiology. J. Biomed. Photonics & Eng. 2016, 2, 020201. 43. Barer, R.; Tkaczyk, S. Refractive Index of Concentrated Protein Solutions. Nature 1954, 173, 821–822. 44. Popescu, G.; Park, Y.; Lue, N.; Best-Popescu, C.; Deflores, L.; Dasari, R. R.; Feld, M. S.; Badizadegan, K. Optical Imaging of Cell Mass and Growth Dynamics. Am. J. Physiol. Cell Physiol. 2008, 295, C538–C544. 45. Li, H.; Zhang, Q.; Yap, C. C. R.; Tay, B. K.; Edwin, T. H. T.; Olivier, A.; Baillargeat, D. From Bulk to Monolayer MoS2: Evolution of Raman Scattering. Adv. Funct. Mater. 2012, 22, 1385–1390. 46. Bang, G. S.; Nam, K. W.; Kim, J. Y.; Shin, J.; Choi, J. W.; Choi, S. Y. Effective - 29 -



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Liquid-Phase Exfoliation and Sodium Ion Battery Application of MoS2 Nanosheets. ACS Appl. Mater. Interfaces 2014, 6, 7084–7089. 47. Coehoorn, R.; Haas, C.; De Groot, R. A. Electronic Structure of MoSe2, MoS2, and WSe2. II. The Nature of the Optical Band Gaps. Phys. Rev. B 1987, 35, 6203–6206. 48. Benavente, E.; Santa Ana, M. A.; Mendizábal, F.; González, G. Intercalation Chemistry of Molybdenum Disulfide. Coordin. Chem. Rev. 2002, 224, 87–109. 49. Ellman, G. L. Tissue Sulfhydryl Groups. Arch. Biochem. Biophys. 1959, 82, 70–77. 50. Pompella, A.; Visvikis, A.; Paolicchi, A.; De Tata, V.; Casini, A. F. The Changing Faces of Glutathione, a Cellular Protagonist. Biochem. Pharmacol. 2003, 66, 1499– 1503. 51. Ukeda, H.; Maeda, S.; Ishii, T.; Sawamura, M. Spectrophotometric Assay for Superoxide Dismutase Based on Tetrazolium Salt 3′-{1-[(Phenylamino)-carbonyl]-3, 4-tetrazolium}-bis (4-methoxy-6-nitro) Benzenesulfonic Acid Hydrate Reduction by Xanthine–Xanthine Oxidase. Anal. Biochem. 1997, 251, 206–209. 52. Hummers Jr., W. S.; Offeman, R. E. Preparation of Graphitic Oxide. J. Am. Chem. Soc. 1958, 80, 1339–1339. 53. Lyon, D. Y.; Alvarez, P. J. J. Fullerence Water Suspension (nC60) Exerts Antibacterial Effects via ROS-Independent Protein Oxidation. Environ. Sci. Technol. 2008, 42, 8127–8132. 54. Vecitis, C. D.; Zodrow, K. R.; Kang, S.; Elimelech, M. Electronic-StructureDependent Bacterial Cytotoxicity of Single-Walled Carbon Nanotubes. ACS Nano 2010, 4, 5471–5479. 55. Kang, S.; Herzberg, M.; Rodrigues, D. F.; Elimelech, M. Antibacterial Effects of Carbon Nanotubes: Size Does Matter! Langmuir 2008, 24, 6409–6413. - 30 -


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56. Kang, S.; Mauter, M. S.; Elimelech, M. Physicochemical Determinants of Multiwalled Carbon Nanotube Bacterial Cytotoxicity. Environ. Sci. Technol. 2008, 42, 7528–7534. 57. Kang, S.; Pinault, M.; Pfefferle, L. D.; Elimelech, M. Single-Walled Carbon Nanotubes Exhibit Strong Antimicrobial Activity. Langmuir 2007, 23, 8670–8673. 58. Liu, S.; Ng, A. K.; Xu, R.; Wei, J.; Tan, C. M.; Yang, Y.; Chen, Y. Antibacterial Action of Dispersed Single-Walled Carbon Nanotubes on Escherichia Coli and Bacillus Subtilis Investigated by Atomic Force Microscopy. Nanoscale 2010, 2, 2744–2750. 59. Hui, L.; Piao, J.-G.; Auletta, J.; Hu, K.; Zhu, Y.; Meyer, T.; Liu, H.; Yang, L. Availability of the Basal Planes of Graphene Oxide Determines Whether It Is Antibacterial. ACS Appl. Mater. Interfaces 2014, 6, 13183–13190. 60. Mangadlao, J. D.; Santos, C. M.; Felipe, M. J. L.; Leon, A. C. C.; Rodrigues, D. F.; Advincula, R. C. On the Antibacterial Mechanism of Graphene Oxide (GO) Langmuir-Blodgett Films. Chem. Commun. 2015, 51, 2886–2889. 61. Akhavan, O.; Ghaderi, E.; Esfandiar, A. Wrapping Bacteria by Graphene Nanosheets for Isolation from Environment, Reactivation by Sonication, and Inactivation by Near-Infrared Irradiation. J. Phys. Chem. B 2011, 115, 6279–6288. 62. Perreault, F.; de Faria, A. F.; Nejati, S.; Elimelech, M. Antimicrobial Properties of Graphene Oxide Nanosheets: Why Size Matters. ACS Nano 2015, 9, 7226–7236. 63. Liu, P. Y.; Chin, L. K.; Ser, W.; Ayi, T. C.; Yap, P. H.; Y.; Bourouina, T.; LeprinceWang. Real-Time Measurement of Single Bacterium’s Refractive Index Using Optofluidic Immersion Refractometry. Procedia Eng. 2014, 87, 356–359. 64. Khan, S.; Pierce, D.; Vale, R. D. Interactions of the Chemotaxis Signal Protein CheY - 31 -



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with Bacterial Flagellar Motors Visualized by Evanescent Wave Microscopy. Current Biol. 2000, 10, 927–930. 65. Balaev, A. E.; Dvoretski, K. N.; Doubrovski, V. A. Refractive Index of Escherichia Coli Cells. In Optical Technologies in Biophysics and Medicine III, Proc. SPIE, International Society for Optics and Photonics 2002, 4707, 253–260. 66. Bang, G. S.; Cho, S.; Son, N.; Shim, G. W.; Cho, B. K.; Choi, S. Y. DNA-Assisted Exfoliation of Tungsten Dichalcogenides and Their Antibacterial Effect. ACS Appl. Mater. Interfaces 2016, 8, 1943-1950. 67. Oláh, A.; Hillborg, H.; Vancso, G. J. Hydrophobic Recovery of UV/Ozone Treated Poly (Dimethylsiloxane): Adhesion Studies by Contact Mechanics and Mechanism of Surface Modification. Appl. Surf, Sci. 2005, 239, 410-423. 68. Song, J.; Tranchida, D.; Vancso, G. J. Contact Mechanics of UV/Ozone-Treated PDMS by AFM and JKR Testing: Mechanical Performance from Nano-to Micrometer Length Scales. Macromolecules, 2008 41(18), 6757-6762.

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Figure 1. Schematics and surface topographies of nanosheet films visualized by atomic force microscopy (AFM). (A) Schematic of GO, MoS2, and the GO-MoS2 nanocomposite film. (B) AFM images of GO, (C) MoS2 nanosheets, and (D) the GO-MoS2 nanocomposite surface on the SiO2/Si. Scanning area of AFM images is 5 µm × 5 µm for each sample, and the corresponding AFM height profiles are shown in the insets of Figure 1B–D. Scale bars: 1 µm.

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Figure 2. Microbial viability measurements and scanning electron microscopy (SEM) images of E. coli. (A) Inactivation of E. coli bacteria came in contact with the GO, MoS2, and the GO-MoS2 nanocomposite film for 3 h. Loss of viability was measured using the colony counting method. (B) Time-dependent antibacterial activity of the GO-MoS2 nanocomposite film. Bacterial suspensions were exposed to the nanocomposite film for 2, 4, and 6 h, and recollected for overnight incubation on LB agar plates. Error bars represent the standard deviation. (C–F) - 34 -


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SEM images of E. coli measured at magnifications of 104 and 2×104 in (C) and (D-F), respectively, after 3-h incubation. Control bacterial cells were shown in (C) and (E) having a typical rod-shape with no membrane destruction. The morphology of E. coli cells contacted with the GO-MoS2 nanocomposite surface is shown in (D) and (F). The SEM images reveal the destruction of bacterial cell membranes and cytoplasmic leakage after having contacts with the GO-MoS2 nanocomposite film. Scale bars: 2 µm in (C), 1 µm in (D-F).

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Figure 3. Oxidative stress mediated by GO, MoS2, and the GO-MoS2 nanocomposite. (A) ROS-dependent oxidative stress at exposure times of 2–6 h. The production of superoxide anion (O2•−) by each nanosheet dispersion was monitored using XTT tests, where XTT alone was used as a negative control. (B) In vitro glutathione oxidation using Ellman’s assay. The loss of glutathione was measured after incubation with GO, MoS2, and GO-MoS2 nanocomposite dispersions for 2-6 h. H2O2 (1 mM) was used as a positive control. Error bars indicate the standard deviation.

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Figure 4. X-ray photoelectron spectroscopy (XPS) of the deconvoluted C(1s) core level peaks in (A) GO and (B) the GO-MoS2 nanocomposite film. (C) The intensity ratios of different functional groups: (C=O, C-OH, and O=C-OH) to (C-C, C=C, and C-H bonds) in the C(1s) region of GO and the GO-MoS2 nanocomposite film.

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Figure 5. Antibacterial activity of the GO-MoS2 nanocomposite film on the transparent silica glass. (A) Loss of viability of E. coli cells came in contact with GO-MoS2 nanocomposite surface on the SiO2/Si and silica glass. Error bars represent the standard deviation. (B) Transmittance spectra of bare glass and the GO-MoS2 nanocomposite film on the glass. Corresponding photographs are shown in the insets.

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Figure 6. Time-lapse measurement of the 3D RI tomogram of bacteria and changes in dry mass and cell volume of live bacteria. (A) Representative cross-sectional images of 3D RI tomograms of a bacterial cell came in contact with 2D nanocomposite surface for 90 min on the focal plane. Upper and lower images indicate the 3D RI tomogram of a bacterial cell along x-z axes and x-y axes over time, respectively. Scale bar: 2 µm. (B) 3D rendered RI distribution of the bacterial cell. Arrows in (A) and (B) indicate the obviously disrupted area of the bacterial membrane. (C) Quantification of relative changes in the dry mass and (D) cell volume of bacteria over time. The vertical lines indicate the standard deviation. Both dry mass and cell volume of bacteria decreased after interaction with 2D nanocomposite surface revealing the degradation of cell membrane and - 39 -



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loss of intracellular components. The asterisks indicate the statistical significance with different number of asterisks showing statistical differences (p < 0.05).

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Table of Contents Graphic

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Antibacterial Activities of Graphene OxideâMolybdenum Disulfide

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